Pyrazinamide (PZA) is an important frontline tuberculosis (TB) drug that is involved in shortening the TB therapy. PZA is an unconventional and paradoxical drug. Despite its powerful sterilizing activity in vivo and its importance in achieving the short course therapy, PZA has no activity against M. tuberculosis under normal culture conditions but is active at acid pH, and PZA kills nongrowing bacilli more effectively than actively growing bacilli. The recent interest to develop new TB drugs that can shorten the therapy has highlighted the importance to understand the mode of action of PZA. Although PZA has been used in clinical treatment of TB since 1952 -the same year as INH, its mode of action is the least understood of all TB drugs because of the paradoxical nature of PZA. Work from this laboratory supported by this application has resulted in a number of significant findings about the mode of action of PZA. Based on these observations, we hypothesize that pyrazinoic acid (POA), the active form of PZA, targets the membrane and interferes with membrane energetics. In this application, we propose the following specific aims to further our understanding of this important TB drug: (1) To investigate the effect of factors that affect membrane energy metabolism on the activity of PZA/POA against M. tuberculosis. These factors include: low oxygen, weak acids, starvation, respiratory chain enzymes and aconitase (2) To assess the effect of enhancers of PZA activity on further improving the sterilizing activity of PZA against M. tuberculosis in vivo in mice, (3) To identify new mechanisms of PZA action and resistance by comparing the gene expression profiles of resistant strains without pncA mutations with sensitive strain in response to PZA. These studies will improve our understanding of mechanisms of PZA action and resistance and should have implications for the design of new antituberculosis drugs that can shorten the TB therapy.